Descriptions of a new genus and a new species, Grylloprimevala jilina (Grylloblattidae) from China

Abstract We erected a new genus (Grylloprimevala Zhou & Ren gen. nov.) and defined a new species (Grylloprimevala jilina Zhou & Ren sp. nov.) from a natural cave in the primeval forest of Jilin Province, China, according to the morphological, behavioral, and molecular evidence. Grylloprimevala gen. nov. is distinguishable from other genera of Grylloblattodea primarily by morphological characters, including the slightly concave posterior margin of the pronotum and no poorly sclerotized zones, six intramarginal and nine intermarginal setae on the cervical sclerite, one tooth on the lacinia, no pulvilli on tarsal segments, and a symmetrical epiproct with a pointed triangular and middle‐depressed median projection on the posterior margin. Based on the morphological features mentioned above, we further identified a new species, G. jilina sp. nov. At the aspect of behavior, G. jilina sp. nov. displays the typical characteristics of troglobites, including degraded visual senses, developed body surface sensors, and predation between individuals. Furthermore, molecular phylogenetic analyses also supported the morphological delimitation of G. jilina sp. nov. due to the separate clade of G. jilina sp. nov. Our results provide materials for the determination and conservation of Grylloblattodea in China.

Grylloblattidae are extremely rare in China. To date, only two specimens have been captured (Bai et al., 2010). In 1987, Mr. Shu-Yong Wang collected the first specimen of a grylloblattid in the Changbai Mountains (Jilin Province, China), and this specimen was named Galloisiana sinensis Wang. The author provided a detailed morphological description of the specimen and showed that despite many similarities with G. nipponensis Caudell & King, there were many differential features such as a concave shape in the middle of the hind margin of the pronotum, fewer antennal segments, anterior legs with slender femora, more and denser setae on the inner margin beneath, and a different shape of the epiproct. The author also concluded that the species differed from Grylloblattina kurentzovi Pravdin & Storzhenko in that the middle of the hind margin of the pronotum protruded posteriorly in a horn shape; the species differed from the two species Namkungia biryongensis Namkung and Galloisiana kosuensis Namkung collected from caves in North Korea in that the two species had degenerated compound eyes (Kim & Lee, 2007;Wang, 1987). In 2009, Mr Ke-qing Song collected the second grylloblattid at Lake Akekule (Lake Bai) (Wang, 1999). In 2010, a team led by Bai Ming in the Institute of Zoology, Chinese Academy of Sciences, conducted a systematic study of the second new species of Grylloblattidae discovered in China (G. cheni Bai, Wang & Yang). In addition to providing a distribution map and a key to the species of Grylloblattella, the authors investigated the thorax, especially the pronotum, of extant and extinct Grylloblattodea using geometric morphometric analysis. The results showed that the high diversity of extinct Grylloblattidae may reflect their diverse habitats and niches, and the diversity of modern Grylloblattidae could be explained by synapomorphy or convergent evolution. The authors also showed that most fossil Grylloblattidae had a clearly longer meso-and metathorax than prothorax, whereas modern Grylloblattidae have a shorter metathorax than prothorax, a phenotype related to the loss of wings and with the associated muscle reduction and changes in the thoracic skeleton system. Finally, the authors described some possible threats to the survival of Grylloblattidae and provided some suggestions for their conservation (Bai et al., 2010).
In summary, there are few studies on Grylloblattidae; their geographical distribution and phylogenetic status are significantly understudied, and their life history and biological characteristics are poorly understood. In particular, Grylloblattidae are extremely understudied in China, and type specimens are very scarce. In this study, we systematically investigated an extremely important new grylloblattid found near Ji'an City, Jilin Province, China. Morphological and molecular data were obtained to explore the phylogenetic status of Grylloblattidae and to confirm the phylogenetic relationships of the new genus with the other five genera and other insects. This paper adds a new genus to Grylloblattodea, provides important material for the determination of the phylogenetic status of Grylloblattodea, and enriches the morphological and molecular information for the study of Grylloblattodea evolution. This study is of significance for the origin and geographic dispersal of Grylloblattidae. In the future, industrial development, human activities, and global warming may threaten the reported and undiscovered Grylloblattidae, and this research will be relevant to the conservation of Grylloblattidae.

| Sampling
Specimens were found in good condition (vivid coloration, robust exoskeleton, and free movement) in a dark area of a natural cave in a primeval forest near Ji'an city, Jilin Province, China, located at 42°11′10.32″N, 123°43′22.75″E, 132 m above sea level (1 in Figure 1a). The location is a karst cave with no direct sunlight. At the bottom of the cave, there are small pools and mounds of earth and rocks, and stalactites on the roof and walls of the cave drip water year-round. The cave temperature is 16.4°C, and the cave humidity is 85.2%. The collected rock piles were located 12 m from the entrance and 2.5 m below the surface (2 in Figure 1a). We collected specimens from May 19, 2020, to May 22, 2020, and June 19, 2021, to June 22, 2021. The new species has only been found in this natural cave in the primeval forest near Ji'an city, Jilin Province, China.
All specimens were collected by hand sampling. We used entomological tweezers and scoopulas (stainless steel spatulas used in chemistry) to protect the biological material.
Specimens were placed in insect boxes containing soil collected from the type locality. The insect box was temporarily placed in an incubator, and an ice pack was placed on either side of the insect box. The incubator was taken back to the laboratory.

| Observations
The specimens were placed in a 40 cm × 30 cm × 30 cm ecological rearing tank. The soil collected from the type locality was placed in the tank, and a thermometer and hygrometer were also put into the tank. The ecological rearing tank was placed in an incubator with a consistent temperature of 16.4 ± 1°C and humidity of 85.2 ± 1%. The incubator was always kept in the dark.
The observation experiment lasted for 6 days and was divided into three stages. On the first and second days, we placed Diplura (as food) caught in the same cave with the specimens in the tank. From the third to the sixth day, Grylloblattidae were not fed. Infrared cameras (Sony Digital Handycam HDR-CX760E) were used to record the conditions in the ecological rearing tank during the three periods of 07:00-09:00, 13:00-15:00, and 19:00-21:00 every day. The focal length of the lens was adjusted when the camera was set up to keep the pictures clear and the entire tank within the camera's view. The video data were then numbered, named, and saved in the computer.
Two researchers were assigned to play the video at normal speed (50 frames per second) and observe the recorded behaviors of the specimens.

| Morphological analysis
This report is based on the study of the adult male specimens, described here as representing a new species. The specimens were identified using species descriptions of Grylloblattidae in Storozhenko (1988). All specimens in the type series were preserved in 70% ethanol. One holotype and two paratypes were collected.
Voucher specimens and a research collection were deposited at Jilin Provincial Key Laboratory of Animal Resource Conservation and Utilization (Northeast Normal University, Changchun, China).
Measurements are in millimeters unless noted otherwise.

| Phylogenetic analysis
Genomic DNA was extracted from the legs of the two paratypes by the phenol chloroform method for subsequent genetic analysis.

Beijing, China) and then transferred into Trans-T1 Phage Resistant
Chemically Competent Cells (CD501-02; Transgen Bio, Beijing, China). The monoclonal colonies were selected by overnight culture at 37°C followed by sequencing and splicing with general primers M13 (Sangon Sequencing) ( Table 1). The sequences of other Grylloblattidae samples used to construct the phylogenetic tree were obtained from the NCBI database. After sequence alignment with PhyloSuite (Zhang et al., 2020), the best nucleotide substitution model was calculated by ModelFinder, and the maximum likelihood tree was constructed. The constructed evolutionary tree was im-

| Diagnosis of the new genus
Posterior margin of pronotum slightly concave, without poorly

| Composition
The genus is monotypic.

| Etymology
The new genus was named after the primitive features.

| Type locality
Located in a dark area of a natural cave in a primeval forest near Ji'an city, Jilin Province, China.  (2004) COII R-lys GAGAC CAG TAC TTG CTT TCA GTCATC (6 in Figure 1c). The tongue is shaped like an oval bowl and membranous; the base is slightly narrower than the distal; and the surface is slightly sclerotized and glabrous (7 in Figure 1c). Mandibles are developed (length = 1.14 mm, width = 0.69 mm), symmetrical, and highly sclerotized. There are two teeth in the distal part and one tooth on the inner margin, and there is no grinding area (9-10 in Figure 1c).

| Type material
The maxillae are larger (length = 2.09 mm, width = 0.61 mm). The distal part extends past the top of the mandibles. They are composed of the cardo, stipes, lacinia, galea, and maxillary palpus. The structure belongs to Orthoptera (11-13 in Figure 1c). The galea is sickle-shaped (length = 0.88 mm, width = 0.11 mm). The distal part is strongly curved and weakly sclerotized and has a semicircular shape with serrated edges on the inner top and scattered dark brown setae on the inner margin (12 in Figure 1c). The lacinia is similar in shape to the galea (length 0.98 mm, width 0.21 mm). It is curved, dark in color, and strongly sclerotized. There is one preapical tooth, the base does not contain teeth, there is one row composed of four dark brown setae between the base and apical teeth (11 and 13 in Figure 1c).
The maxillary palp has five palpomeres and is 1.98 mm (0.14 mm + 0 .16 mm + 0.48 mm + 0.48 mm + 0.72 mm) long. The first palpomere is the shortest, and the second is one-third the length of the third. The length of the third is the same as that of the fourth; and the fifth is the longest (1.5 times longer than the fourth) and is densely covered with fine, short, light brown hairs (12 in Figure 1c). The labium has a pair of labial palps, a pair of paraglossae and a pair of smaller glossae (8 in Figure 1c). There are three segments in the labial palps, with a total length of 0.97 mm (0.23 mm + 0.25 mm + 0.49 mm). The first and second segments contain a few dark brown setae, and the third is covered with many fine, short, light brown hairs (8 in Figure 1c).
There is a row of dark brown setae on the outer margin of the paraglossae and glossae without setae (8 in Figure 1c).
The pronotum is 1.13 times as long as it is wide (length = 2.23 mm, width = 1.97 mm) and is quadrilateral. The anterior part of the pronotum has a deep transverse sulcus, is elongated anteriorly, lacks poorly sclerotized zones, and has some dark brown setae on its anterior margin and scattered dark brown setae on its dorsum. The longitudinal suture is continuous and long (14 in Figure 1d). The mesonotum is 1.09 times as long as it is wide (length = 1.32 mm, width = 1.21 mm), has scattered dark brown setae on its dorsum, and lacks a longitudinal suture (14 in Figure 1d). The metanotum 1.95 times as wide as it is long (length = 0.93 mm, width = 1.81 mm), has scattered dark brown setae on the posterior margin, and lacks a longitudinal suture (14 in Figure 1d). The posterior margin of the pronotum is slightly concave, and the mesonotum and metanotum are broadly rounded and clearly concave in the posterior part.
The cervical sclerites is about twice as long as it is wide (length = 1.12 mm, width = 0.56 mm), is broadly triangular and elongated anteriorly, and has six dark brown setae on the inner margin and nine dark brown setae on lateral margin (15 in Figure 1d).
The basisternum of the prothorax is 1.38 times as wide as it is long (length = 0.74 mm, width = 1.02 mm), is triangular, and has a long median suture and four dark brown setae on the anterior part (15 in Figure 1d). The basisternum of the mesothorax is 1.7 times as wide as it is long (length = 0.56 mm, width = 0.96 mm) and has a median suture originating posteriorly and many scattered setae (15 in Figure 1d). The basisternum of the metathorax is about 2.67 times as wide as it is long (length = 0.36 mm, width = 0.96 mm) and has a short median suture originating posteriorly and many scattered setae (15 in Figure 1d).
There are three pairs of legs, including strong forelegs (7.81 mm), and elongated midlegs (7.26 mm) and hindlegs (8.27 mm) (19-21 in Figure 1e). The coxa is large and strong with a distinct rib. The femur of the foreleg is thick, is about 2.8 times as long as it is wide (length = 2.71 mm, width = 0.97 mm), and has a smooth dorsum and has a pair of slightly curved symmetrical apical processes, a pair of oval paraprocts, and an epiproct (17 in Figure 1d). The epiproct of the male is symmetrical with an acutely pointed triangular median projection on the posterior margin and a median depression (16 in Figure 1d).
The cercomeres (3.11 mm) (24 in Figure 1f) have nine segments and are filiform. The lengths of the first and second are equal, length gradually increases from the third to ninth, and the last section is elongated. The cerci is hollow; there are a small number of dark brown setae at the end of each segment, and there are long, thick, dark brown setae at the end of the last segmentare.
The coxopodites are asymmetrical. The right coxopodite is longer than it is wide and sclerotized (length = 0.48 mm, width = 0.32 mm), and the inner side and the basal segment are narrow. The left coxopodite is wider than it is long (length = 0.29 mm, width = 0.36 mm), is membranous, has a strong curved scoop-like shape. The subgenital plate is wider than it its long, and the distal edge is hardened (18 in Figure 1d).

| Behavior
According to the observations, G. jilina sp. nov. often stop to clean their antennae, legs, and cerci with their mouthparts when crawling in order to keep their surface sensors clean and sensitive. As individuals crawl, their antennae vibrate in the air and touch the soil and obstacles to discern direction and to search for food. When two individuals of the same species meet, they sometimes gather in one place and constantly touch each other's antennae or body for a period of time to complete recognition and information communication. Sometimes when the antennae of two individuals come into contact, both insects quickly flee. This constant contact and rapid escape occur without regularity. After placing Anisuracampa ywangana Sendra & Komerički (Diplura) collected in the same cave as the individual in the rearing tank, the individual was found to prey on the diplurans. When there were no other insects in the feeding tank, the individual used insect residues and other organic matter in the soil as food. After the insect residues and other organic matter that could be found in the soil and the diplurans were eaten, individuals began to prey on each other, but they did not prey on each other every time they met, and their walking power decreased significantly after satiation. The insects were usually stationary in one place, crawled a short distance after being disturbed, and then returned to a stationary state.

| Phylogenetic relationships
A maximum likelihood tree was constructed based on the joint dataset of 18 s, 28 s, H3, 12 s, 16 s, and COII genes (Figure 2). Species of Mantophasmatodea, Orthoptera, Zygentoma, Microcoryphia, and Blattodea were selected as outgroups. The topological structure of the evolutionary tree was consistent with the tree constructed by Jarvis and Whiting (Table S1) (Jarvis & Whiting, 2006). In the molecular phylogenetic tree, all species of Grylloblattidae were grouped into a large clade, supporting the monophyly of Grylloblattidae.
In Grylloblattidae, the genus Grylloblattina first differentiated and formed sister branches with other groups of Grylloblattidae.
Grylloprimevala gen. nov. and Galloisiana formed a sister group first, and further clustered as one clade with Grylloblatta.

| Etymology
The specific epithet was named after the habitat and collection site of the new species from Jilin Province, China.

| Behavior
Our observations on the Grylloblattid individuals revealed that

| Comparisons
Grylloprimevala gen. nov. in this study is different from other known genera previously discovered. nov. without pulvilli on the tarsal segments. Furthermore, the posterior margin of supra-anal plate is symmetrical in Grylloprimevala gen.
In conclusion, based on the morphological data, it can be seen

| Ecological environment of distribution area
Almost all Grylloblattidae live in an environment characterized by low temperature throughout the year, snow coverage, and high humidity (Kamp, 1963(Kamp, , 1979Vrsansky et al., 2001). As temperatures become near zero, Grylloblattidae are active during the night, and they usually climb to snowfields after sunset to forage and hide in dark areas such as rock crevices during the day (Mann et al., 1980;Schoville, 2010). The optimal living temperature of Grylloblattidae is between 0 and 1°C, and mortality increases significantly above 16°C; they rarely survive at temperatures below −6.2°C or above 20.5°C (Edwards, 1982;Henson, 1957). Although little is known about the mechanism of action of temperature on their activities, a study of G. jilina sp. nov. in the laboratory showed that they preferred 16.4 ± 1°C, survived between 0 and 23°C, and died at temperatures >25°C. In alpine habitats, Grylloblattidae are mostly found on rocky grounds formed by weathering and in basins covered with snow (Schoville, 2010). At low altitudes, Grylloblattidae live in caves/lava tubes, canyons, and rocky river banks (Edwards, 1982 branches and suggest that they may be closely related sister groups.
However, more morphological characteristics and molecular data are needed to verify this hypothesis.

| Conservation
Because the microhabitat of G. jilina sp. nov. is highly specialized and the species is characterized by scarcity, low abundance, impaired migration, and limited geographical distribution, several recent conservation assessments have concluded that G. jilina sp. nov. can be considered as an endangered species and that its survival is threatened. Climate change (e.g., global warming, dramatic changes in local precipitation, changes in snow cover, and sustained increase in mean annual temperature), anthropogenic damage, and landscape change will directly affect the habitat of G. jilina sp. nov. and lead to its extinction.
Currently, several important measures should be taken to update and improve the conservation and assessment of G. jilina sp. nov. First, an accurate distribution pattern of the new species should be determined before investigation of syntopic occurrence (occurrence of different species at the same locality). Second, long-term investigations for conservation should be conducted at the type specimen distribution site, including the cave where the specimen was found, the ground environment outside the cave, and all caves within a radius of 50 km.
The conservation status we assigned to this novel species provides updated data for the NatureServe database and the IUCN Red List.

| Future research
We currently have sufficient information to determine the activity location, to collect samples, and to monitor the population of G. jilina sp. nov. The challenges in the coming years involve addressing several prominent issues in the systematic study of G. jilina sp.
nov. These include: (1) to thoroughly explore the mechanisms of